Gene transfer efficiency during gestation and the influence of co-transfer of non-manipulated embryos on production of transgenic mice

Litter size of DNA microinjected zygotes is lower than for non-manipulated zygotes. The rate of embryonic and fetal survival in early, mid and late gestation was determined to assess whether DNA integration was responsible for embryonic losses. Also, the effect of including non-microinjected embryos with injected embryos on pregnancy rate and transgenic pup production was determined. In Experiment 1, one-cell embryos from immature CD-1 mice were microinjected with a whey acidic protein promoter-human protein C gene construct. One hour after microinjection embryos were transferred to pseudopregnant recipients (45 transfers of 30 embryos each). Fifteen recipients were sacrificed on day 4, 12 and 18 of gestation and the embryos/fetuses analysed for the transgene. The percentage of embryos or fetuses that were positive for the transgene was not significantly different at any day. However, the number of viable embryos at day 4 was significantly greater than fetuses on days 12 or 18. In addition, a high degree of mosaicism was observed in day 18 fetuses and placentae recovered. In Experiment 2, one-cell embryos from CD-1 mice were microinjected and co-transferred with non-manipulated embryos (C57BL/6). Pregnancy rate and the total number of pups born were improved by addition of non-injected embryos. However, the number of transgenic mice produced was similar whether non-injected embryos were included or not. There were 32.2% (15/46) transgenic pups when 0 non-injected embryos were transferred compared with 15.1% (13/86) transgenic pups when 4 or 8 non-injected embryos were added to the transfers. In summary, a high degree of embryonic and fetal mortality occurs among microinjected embryos. Furthermore, since the percentage of transgenesis did not change throughout pregnancy, DNA integration does not appear to account for all of the embryonic losses. other factor(s) related to the microinjection procedure may be involved in the embryonic and fetal failure of microinjected embryos. Addition of non-injected embryos, although it increased pregnancy rate and the number of pups born from microinjected embryos, actually decreased the number of transgenic pups obtained per pregnancy.     

Development of a pronuclear DNA microinjection technique for production of green fluorescent protein-expressing bubaline (Bubalus bubalis) embryos

Oocytes from abattoir-derived bubaline (Bubalus bubalis) ovaries were subjected to IVM and IVF; the objective was to develop a pronuclear DNA microinjection technique to produce embryos expressing green fluorescent protein (GFP). The largest proportion (61.2%) of zygotes in which one (1 PN) or two pronuclei (2 PN) were visible was when centrifugation (14,000 x g for 15 min) was done 16 h after insemination. Centrifugation had no adverse effects on cleavage rate, development to morulae/blastocysts, and total cell number of embryos. Piercing the pronuclear but not the plasma membrane reduced (P<0.05) cleavage rate (44.0% vs. 51.0%), without affecting subsequent development. Following microinjection of a GFP-DNA construct, cleavage rate (55.9, 38.9, and 30.9%) and proportion of cleaved embryos that developed to morulae (39.9, 25.6, and 15.5%) and blastocyst stages (22.4, 13.4, and 2.8%) were higher (P<0.05) for non-injected controls than for those injected with buffer alone, which, in turn, were higher (P<0.05) than for those injected with buffer containing 5 microg/mL DNA. The cleavage rate (39.2% vs. 34.8%) and proportion of cleaved embryos that developed to morulae/blastocysts (37.5% vs. 10.9%) were higher (P<0.05) for microinjected zygotes with 2 PN than for those with 1 PN. The cleavage rate and the proportion of cleaved embryos that developed to morulae and blastocysts were higher (P<0.05) following culture of microinjected zygotes in mCR2aa medium (40.7, 32.7, and 9.1%, respectively) compared to those for mSOFaa (33.3, 26.0, and 6.5%, respectively) or after culture in TCM-199+co-culture with buffalo oviductal epithelial cells (31.2, 25.0, and 4.5%, respectively). The proportion of embryos expressing GFP was higher (P<0.01) for 2 PN than for 1 PN zygotes (15.9% vs. 13.7%). Thirty-five embryos expressed GFP; the proportion of mosaic embryos (62.8%) was higher (P<0.01) than of embryos in which all blastomeres expressed GFP (37.2%); eight and two of those embryos developed to the morula and blastocyst stages, respectively.     

Efficiency of transgenic rat production is independent of transgene-construct and overnight embryo culture

The aim of the present work was to study factors affecting the efficiency of transgenic technology in rats. We investigated the possible effects of pronuclear microinjection of buffer or different DNA-constructs on survival and development of rat zygotes in vitro and in vivo as well as the influence of overnight culture of these embryos before transfer into pseudopregnant foster mothers. The survival rate of zygotes and their development to the two-cell and morula stage was not affected by pronuclear microinjection with different DNA-constructs or buffer. However, the development to the blastocyst stage was impaired. Nevertheless, there was no difference in blastocyst development between zygotes injected with DNA-constructs or with buffer. Neither was there a difference in cell number in in vitro cultured blastocysts resulting from pronuclear microinjection of a transgene compared with non-injected controls. The survival rate to term was about 30% irrespective of whether microinjected embryos were transferred immediately after microinjection or after overnight culture in vitro. However, a reduction in the survival to term was observed for non-injected zygotes when they were developed in vitro to the two-cell stage before transfer to a pseudopregnant female. The percentage of transgenic rats that resulted from microinjected zygotes was similar in all groups regardless of the DNA-construct used (2.7-10.0%). In conclusion, the main detrimental factor in the microinjection of rat zygotes is the introduction of solution in the pronucleus. Overnight culture of zygotes between microinjection and oviduct transfer does not decrease the efficiency of transgenic rat generation.     

In vitro and in vivo development of bovine embryos from zygotes and 2-cell embryos microinjected with exogenous DNA

The objectives of these experiments were: 1) to determine an effective culture method for production of transferable bovine embryos following exogenous DNA microinjection; 2) to determine the effect of these methods on the ability of the injected zygotes and 2-cell embryos to develop in vivo; and, 3) to compare development of embryos microinjected as zygotes or 2-cell embryos. DNA fragments encoding bovine growth hormone (bGH), bGH-10Delta6, and a bGH antagonist, bGH-M8 (5) were used. A total of 639 zygotes and 153 2-cell embryos were injected. Zygotes and 2-cell embryos microinjected with bGH-M8 were incubated for 6 days in oviducts of intermediate recipients (rabbits or sheep) or co-cultured in vitro with bovine oviduct epithelial cells. Zygotes and 2-cell embryos microinjected with bGH-10Delta6 were co-cultured in vitro only. The most effective method for the production of transferable bovine embryos following exogenous DNA microinjection was via in vitro co-culturing with bovine epithelial cells. For example, 32.3% of the bGH-M8 and 33.5% of the bGH-10Delta6 microinjected zygotes reached the morula/blastocyst stage while 48.4% and 63.0% of the 2-cell embryos injected with bGH-M8 and bGH-10Delta6, respectively, developed to the morula/blastocyst stage. The percentage of blastocysts obtained for control, non-injected zygotes and 2-cell embryos was 34.5% and 69.6%, respectively. The developmental rate to the morula/blastocyst stage was approximately 20% greater for embryos obtained from microinjected 2-cell embryos relative to microinjected zygotes. However, there was no significant difference in pregnancy rates following transfer of these blastocysts to cow uteri.     

Transgene detection during early murine embryonic development after pronuclear microinjection

The polymerase chain reaction (PCR) technique was used to detect a whey acidic protein (WAP) gene and transgene presence in mouse ova cultured to various stages of development after pronuclear microinjection at the one-cell stage. The PCR technique detected an endogenous 442 bp WAP DNA sequence in 78% of one-cell, 88% of two-cell and 94% of four-cell ova, and in 95% of morulae and 97% of blastocysts. The heterologous WAP-human protein C transgene was detected in 88% of one-cell, 88% of two-cell and 44% of four-cell ova, and in 40% of morulae and 29% of blastocysts. For comparison, the integration frequency for transgenic mouse production using the same DNA construct was 22%. After five days ofin vitro culture, embryos that were either developmentally arrested or fragmented were tested for the presence of the transgene. The injected construct was detected in 83% of arrested one-cell, 85% of arrested two-cell, and 85% of fragmented ova. In culture, only 28% of zygotes microinjected with DNA developed to the blastocyst stage compared to 74% of noninjected zygotes, while 63% of zygotes developed to the blastocyst stage after injection of buffer alone. Pronuclear injection of the transgene at concentrations of 1.5, 15 and 50 g ml^–1 resulted in 28, 11 and 9% development to blastocysts and 29, 86 and 88% transgene detection, respectively. Transgene detection was 85, 96 and 97% in degenerate embryos at the respective doses of DNA. These data show that pronuclear microinjection of the transgene is detrimental to subsequent embryonic development. Also, unintegrated copies of the transgene probably exist at least until the blastocyst stage, and thereafter are degraded to the extent that they can no longer be detected by PCR.     

Increased transgene integration efficiency upon microinjection of DNA into both pronuclei of rabbit embryos

Transgenic rabbits provide a useful biological model for the study of the regulation of mammalian genes. However, transgene integration efficiency has generally been low. Here we present a first attempt to increase the integration rate of exogenous DNA into the rabbit genome, using a double pronuclei microinjection method. Pronuclear stage rabbit embryos were recovered from superovulated NZW females, 19–20 h after hCG injection. About 5 μg/mL of exogenous DNA solution was microinjected either into one pronucleus (single microinjection, SM) or into both pronuclei (double microinjected, DM). The transgene consisted of a 2.5 kb murine whey acidic protein promoter (mWAP), 7.2 kb cDNA of the human clotting factor VIII (hFVIII), and 4.6 kb that of 3′ flanking sequences of the mWAP gene. The in vitro survival of DM embryos to the blastocyst stage was lower than that of SM embryos (68 vs. 89%). Similar results were obtained using EGFP as a control gene construct. However, there was no difference in the percentage of embryos that developed into live offspring using DM (25%) vs. SM (26%). The integration frequency of mWAP-hFVIII into the genome of transgenic rabbits was 3.3% (1/30) upon SM and 8.1% (4/49) at DM (p < 0.05). All founders transmitted the transgene to their offspring in a Mendelian fashion. The SM founder female secreted 87.4 μg/mL rhFVIII in milk, with an activity of 0.594 lU/mL. The DM founder female produced 118 μg/mL rhFVIII, with activity values of 18 lU/mL. This is the first report of transgenic rabbit production using a double microinjection technique. Our preliminary results suggest that this method can increase the efficiency of production of transgenic rabbit founders, giving a higher integration rate than single microinjection.     

Expression of microinjected reporter gene lacZ during first cleavages of rabbit embryos

The growing use of reporter genes in a model transgenic system has been a fundamental approach of biology, but the strategy of transgenic embryo selection prior to transfer to foster mothers may greately increase the efficiency of transgenic livestock production. This study was conducted to assess the possibility of beta-galactosidase (beta-gal)-labeled transgenic rabbit embryo production. Rabbit zygotes were obtained from superovulated females after mating. Zygotes were microinjected into male pronuclei with pCMV-lacZ or SV40-lacZ constructs; while some embryos were co-injected with the scaffold attachment sequences--SAR. Embryos from control non-injected and microinjected groups were cultured in vitro. After 24, 48, 72, or 96 h of culture the embryos were stained with X-gal for beta-galactosidase. Transgenic embryos produced by pronuclear injection showed a discrete pattern of beta-galactosidase expression. The percentage of transgenesis with pCMV-lacZalone was 1.5, but with SAR sequences it increased to 4.2. In the case of SV40-lacZ construct, the efficiency of transgenesis was 2.3% and 4.1%, respectively. The mosaicism was 66.7% for all embryos injected with both constructs with or without SAR. The highest numbers of 100%-transgenic (non-mosaic) embryos were found in the group co-injected with SV40-lacZ and SAR. Transgenesis was seen as early as 24 h after injection, in four-cell embryos. Most of the microinjected embryos showed delayed development as compared with control. It was concluded that lacZ may serve as a reliable reporter for early transgenic embryo selection in order to produce transgenic animals.     

Fate of microinjected genes in preimplantation mouse embryos.

The state of genes microinjected into mouse embryos was followed from the one-cell to the blastocyst stage using the polymerase chain reaction (PCR). Microinjected DNA was detected in all one-, two-, and four-cell injected embryos and in 44% of morula and 26% of blastocysts. Head-to-tail ligation of microinjected genes, a common feature of stably integrated transgene arrays, was detected in all embryos after injection of microinjected genes and occurred irrespective of the structure at the ends of the injected genes. Sensitivity of microinjected DNA to a methylation-dependent restriction endonuclease Dpn I was lost in all embryos by the two-cell stage (24 hr), indicating a change in DNA methylation, independent of transgene integration. Dissociation of blastomeres prior to compaction revealed a mosaic distribution of the microinjected DNA within the embryo and supports the notion that injected genes form a limited number of arrays, which segregate independently until they integrate into the genome or are degraded.     

Pronuclear visibility, development and transgene expression in IVM/IVF-derived porcine embryos

A total of 1550 zygotes was used to assess the timing of pronuclear visibility, embryo development following DNA microinjection, and transgene expression in IVM/IVF-generated porcine embryos. After centrifugation, pronuclei could be seen in 61.6% of zygotes. In 55.3% of these only 1 pronucleus was visible. Pronuclear visibility was highest at 20 h post-insemination. Zygotes were microinjected with 1 of 2 LacZ gene constructs driven by either the SV40 early promoter (pSVON) or the human cytoplasmic beta actin promoter (pbActinLacZ). Development and transgene expression were assessed after either 48 h or 7 d in culture. After 48 h, significantly more zygotes with a single visible pronucleus developed to the 8-cell stage than zygotes in which no pronucleus had been seen (43.0 vs 24.8%), while those with 2 pronuclei were intermediate (31.4%). After 7 d, no difference in development to the morula stage was observed between noninjected control embryos (25.5%) and embryos with 1 (21.0%) or 2 pronuclei (22.5%); however, the proportion of embryos reaching the morula stage in the nonpronuclear group was significantly reduced (9.1%). After 48 h in culture, transgene expression was significantly higher in embryos with 2 pronuclei at the time of injection than in those with 1 (36.4 vs 17.9%). After 7 d in culture, 41.5% of morulae derived from zygotes with 2 pronuclei and 29.97% of thsoe derived from zygotes with 1 pronucleus showed signs of transgene expression. At this stage, significantly more morulae expressed the pbActinLacZ than the pSVON transgene (43.8 vs 25.8%). More than 80% of putative transgenic morulae or blastocysts showed evidence of mosaicism. These results demonstrate that IVM/IVF porcine embryos are able to develop in culture and express a microinjected transgene.     

FLPe functions in zebrafish embryos

To assay the efficiency of the FLP/FRT site-specific recombination system in Danio rerio, a construct consisting of a muscle-specific promoter driving EGFP flanked by FRT sites was developed. FLPe capped RNA was microinjected into transgenic single cell stage zebrafish embryos obtained by crossing hemizygous transgenic males with wild-type females. By 48 h post fertilization (hpf), the proportion of embryos displaying green fluorescence following FLPe RNA microinjection was significantly lower (7.7%; P < 0.001) than would be expected from a cross in the absence of the recombinase (50%). Embryos that retained fluorescence displayed marked mosaicism. Inheritance of the excised transgene in non-fluorescent, transgenic embryos was verified by PCR analysis and FLPe-mediated recombination was confirmed by DNA sequencing. Sperm derived from confirmed transgenic males in these experiments was used to fertilize wild-type eggs to determine whether germline excision of the transgene had occurred. Clutches sired by FLPe-microinjected males contained 0–4% fluorescent embryos. Transgenic males that were phenotypically wild-type produced no fluorescent progeny, demonstrating complete excision of the transgene from their germline. FLPe microinjected males that retained some fluorescent muscle expression produced a small proportion of fluorescent offspring, suggesting that in mosaic males not all germline cells had undergone FLPe-mediated transgene excision. Our results show that FLPe, which is derived from Saccharomyces cerevisiae, is an efficient recombinase in zebrafish maintained at 28.5°C.     

Use of bovine satellite sequences to increase transgene integration by homologous recombination in bovine embryos

Homologous recombination (HR) has proven to be functional in mammalian embryos. The efficiency of the HR process was tested in bovine zygotes in an attempt to increase the frequency of transgene integration using different lengths of a bovine satellite (BS) DNA flanking both ends of a neo gene marker (called BS500, BS250, and BS50) and neo alone as a control. Pronuclear microinjection at 16-19 hr post insemination (hpi) of the BS500, BS250, BS50 or neo fragments at a concentration of 1 ng/microl resulted in an increasingly negative effect on embryo development. Therefore all microinjections were performed at a single molecular concentration (320 x 10(6) molecules/ microl). After microinjection, the embryos were allowed to develop for 6 days followed by morphological and PCR analysis. The HR event was detected by PCR in 13 of the 26 embryos (43%) that developed beyond the 12-cell stage, 7/22 (31%), 9/27 (33%), and 0/25 (0%) with the BS500, BS250, BS50, and neo constructs respectively. The length of BS homology had no effect on transgene integration. However, embryos injected with BS neo constructs had significantly lower development rates than neo injected zygotes (17% more than 16 cells for BS500; 14% for BS250; 16% for BS50 compared to 32% for neo, P < 0.05, 6 replicates). These results demonstrate that BS sequences have a negative effect on embryo development and survival regardless of the amount of DNA injected. The use of HR with highly repetitive genomic sequences is therefore a feasible procedure to produce transgenic bovine embryos.     

The influence of pre- and post-ovulatory insemination on sperm distribution in the oviduct,accessory sperm to the zona pellucida,fertilisation rate and embryo development in sows

The aim of present study was to investigate the influence of pre-compared with post-ovulatory insemination, on the distribution of spermatozoa in the oviduct, the accessory sperm counts on the zona pellucida and early embryonic development. Thirty-six crossbred multiparous sows (Swedish Landrace x Swedish Yorkshire) were artificially inseminated once either at 20-15 h before (group AIB) or at 15-20 h after (group AIA) ovulation by using a pooled semen of two boars. Thereafter, they were randomly allocated to one of five groups: slaughter at 5-6h after AI (group I-AIB), at 20-25 h after ovulation (groups II-AIB and II-AIA), at 70 h after ovulation (groups III-AIB and III-AIA), on day 11 (groups IV-AIB and IV-AIA, first day of standing oestrus=day 1) and on day 19 (groups V-AIB and V-AIA).The plasma levels of oestradiol-17beta and progesterone differed significantly (P相似文献   

Sexing and detection of gene construct in microinjected bovine blastocysts using the polymerase chain reaction

We present a polymerase chain reaction (PCR)-based procedure for rapid bovine embryo sexing and classifying embryos for the presence of exogenous DNA. Fourteen bovine blastocysts microinjected with gene construct DNA at the pronuclear stage were divided into quarters and subjected to amplification with construct-specific and sex gene-specific (ZFY/ZFX) primers in the same initial PCR reaction. Blastocysts carrying microinjected construct DNA could be identified by the presence of construct-specific PCR product in approximately 4 h. Approximately half of the microinjected and two of 16 non-microinjected blastocysts typed PCR-positive for the construct DNA. Owing to erroneous amplifications in the two non-microinjected control blastocysts, and the inability of the system to distinguish integrated from non-integrated copies of the microinjected construct, the number of construct-positive blastocysts determined in our assay most likely overestimates the number of true transgenic embryos. Nevertheless, using this assay, we were able to determine that approximately half of the microinjected embryos were negative for the transgene construct and thus could be eliminated from transfer to a recipient cow. Embryo sexing was achieved in less than 6 h by restriction fragment length polymorphism analysis of nestedZFY/ZFXPCR products reamplified from initial PCR reactions. In 11/14 microinjected blastocysts all sections assayed unambiguously as the same sex. In one embryo, only one section was analysed, while two other blastocysts whowed some discrepancies of sexing results between the sections analysed. The approach employed here to determine the sex and presence of microinjected construct DNA in bovine preimplantation embryos is rapid, accurate among different sections of an embryo and can be used to increase the efficiency of current transgenic cattle production procedures.     

Enhancing the rate of recovery and quality of the embryos in repeat breeding cows by using a GnRH analogue injection at mid-luteal phase prior to breeding

Nonsurgical embryo collections were made 11 days after AI from a total of 101 repeat breeding dairy cows over two complete years. The 101 cows were randomly assigned to 1) control (n =52) or 2) treated (n= 49) groups. After a third unsuccessful AI or more (with previous regular intervals between AI), no insemination took place at the following heat. Then at Day 12 (day 0 = estrus), the former received 5 ml injection i.m. of a saline solution (placebo) and the treated group was injected with 20 μg of a GnRH analogue (Buserelin, Hoe 766). The cows were inseminated on the following estrus with semen of average or above fertility.The GnRH analogue-treated group had a much higher recovery rate of embryos than the controls (65% vs 32%, p<0.005). Similarly the treated group had 91% good embryos (fertilized ova with the appropriate stage of development) out of those recovered; this rate was much higher than that of the controls (57%; P<0.05), resulting in 59% of good embryos per collected treated cows versus 19% in the controls (p<0.001).Practically, these data prove the benefits of this GnRH analogue therapy at mid-luteal phase before AI in nonsuperovulated repeat breeding bovines.     

The effect of timing of laparoscopic insemination in superovulated ewes, with or without sedation, on the recovery of embryos, their stage of development and subsequent viability

Twenty ewes were used as donors in a 2x2 factorial design experiment to investigate the effects of two different insemination times (48 vs 60 h after pessary withdrawal), with or without sedation, on the ovum recovery rate 5 d after insemination, the proportion of transferable embryos recovered, and the subsequent survival rate of embryos transferred to recipients. The ovum recovery rate following intauterine insemination at 48 h after progestagen pessary withdrawal was 63.8 and 53.4% for sedated and nonsedated control ewes, respectively. Following intrauterine insemination at 60 h the corresponding values for sedated and control ewes were 72.6 and 73.9%, respectively. The proportion of transferable quality embryos recovered was not affected by sedation but was improved by insemination at 48 h rather than 60 h after pessary withdrawal (100 vs 35.4%). Embryo survival following laparoscopic transfer to recipients from donor ewes inseminated at 48 h, with or without sedation was 38.8% (7 18 ) and 50% (7 14 ), respectively. Following intrauterine insemination of the donors at 60 h, the survival rate in recipients was reduced for embryos transferred from both the sedated and control ewes to 6.25% (1 16 ) and 36.3% (4 11 ). It is concluded that delaying the timing of intrauterine insemination relative to pessary withdrawal and the use of acepromazine maleate as a sedative at the time of insemination are deleterious to embryo development and subsequent viability.     

Low levels of chimerism in rabbit fetuses produced from preimplantation embryos microinjected with fetal gonadal cells

The potential pluripotency of rabbit fetal germ cells has been investigated by using them to make chimeric embryos. Gonial cells, isolated from enzyme-dispersed male and female transgenic fetal rabbit gonads of 18–22 days gestation, were microinjected in groups of about 10 into 640 nontransgenic rabbit embryos between the two-cell and expanded blastocyst stages. Injections were made with primary isolations of gonial cells, within 48 hr of their collection. The injected embryos were transferred, with or without non-injected control embryos, into 49 recipient rabbits. Tissues from 159 resulting fetuses, implantation sites, and a few liveborn young were examined by PCR analysis for the two transgenes used (α-antitrypsin or luciferase). The overall pregnancy rate (about 80%) was not affected by the stage of development of the embryo injected, nor by co-transfer of control embryos. The survival rate of injected embryos (18% overall, 23.6% in pregnant recipients) was almost identical to that of 243 control embryos. Chimerism was detectable in tissues produced from 4 of 159 (2.5%) of the injected embryos, all four of which had been injected at the 8 to 16-cell stage. This low rate of success indicates that, although passage of rabbit gonial cells is not an absolute requirement for pluripotency, further investigation should pay particular attention to improving culture conditions with a view to deriving EG cell lines. © 1996 Wiley-Liss, Inc.     

Detection of microinjected genes in bovine preimplantation embryos with combined DNA digestion and polymerase chain reaction

We have developed a simple digestion-polymerase chain reaction (PCR) assay for a simultaneous transgene detection and sexing of pronucleus-injected bovine preimplantation embryos. Bovine embryos were microinjected with dam-methylated gene construct and cultured in vitro for 6–7 days after the injections. The developed blastocysts and compact morulae were bisected and the embryonic biopsies representing mainly trophoblasts were subjected to the digestion-PCR, while the biopsied embryos remained in culture. Embryonic DNA was released with proteinase K and the samples were digested with a Dpnl-Bal31 mixture before the PCR amplification of the transgene, bovine αS1-casein, and bovine Y-chromosome fragments in the same reaction. The whole assay from biopsy to electrophoresis took less than 6 hr. The digestion removed up to 50 fg of dam-methylated transgene copies (unintegrated or contaminants) and also a few hundred copies of contaminating PCR products from the embryonic samples. The digestion-PCR assay eliminated all transgene contaminations from noninjected blastocysts, which were exposed to the microinjection DNA during the stay in injection chambers, and reduced the amount of transgene-positive embryos among pronucleus-injected blastocysts as compared with unmodified PCR. Analysis of 486 microinjected bovine embryo biopsies in 13 separate experiments revealed that we were able to sex 398 (82%) of the biopsies and 77 (19%) of the biopsies were scored as transgene positive and 57 (14%) as transgene questionable. Upon reanalysis of 41 of the biopsied embryos, 38 (93%) of the embryos were observed to be transgene negative and 2 questionable in both assays and uneven distribution of transgene copies was observed in one embryo. The results from sexing were in accordance with biopsies and remaining embryos in 38 (93%) of the embryos. © 1996 Wiley-Liss, Inc.     

Embryo production from superovulated Holstein-Friesian dairy heifers and cows after insemination with frozen-thawed sex-sorted X spermatozoa or unsorted semen

The aim of this study was to evaluate embryo production in superovulated Holstein-Friesian dairy heifers and cows inseminated with either X-sorted spermatozoa (2 million/dose) or unsorted semen (15 million/dose). Experiment 1 at the research farm involved eight heifers, six cows and semen of one Holstein bull. All transferable embryos were diagnosed for sex. Experiment 2 included embryo collections on commercial dairy farms: X-sorted spermatozoa from three Holstein bulls were used for 59 collections on 28 farms and unsorted semen from 32 Holstein bulls were used for 179 collections on 79 farms. Superovulations were induced by eight declining doses of FSH (total of 12 ml for heifers and 19 ml for cows) starting on days 8-12 of the estrus cycle. Inseminations began 12h after the onset of estrus and were performed two to four times at 9-15 h intervals. Low-dose X-sorted inseminates were deposited into uterine horns and unsorted semen was placed into the uterine body. In Experiment 1, on average 70.3 and 75.0% of embryos recovered from heifers, and 48.4 and 100% of embryos recovered from cows were of transferable quality in X-sorted and unsorted groups, respectively. The proportion of transferable female embryos produced approximately doubled when insemination was with X-sorted spermatozoa compared to insemination with unsorted semen (heifers 96.4% versus 41.1%; cows 81.1% versus 39.8%). In Experiment 2, estimated 53.9 and 65.5% of embryos recovered from heifers, and 21.1 and 64.5% of embryos recovered from cows were of transferable quality in X-sorted and unsorted groups, respectively. Proportions of unfertilized oocytes were 21.1 and 10.6% for heifers and 56.0 and 14.4% for cows in X-sorted and unsorted groups, respectively. Consequently, cows inseminated with X-sorted spermatozoa produced significantly smaller proportions of transferable embryos (p<0.005) and significantly larger proportions of unfertilized oocytes (p<0.001) than those inseminated with unsorted semen. Proportions of quality 1 or degenerated embryos were similar for the two treatments in both heifers and cows. Within treatments, bulls did not significantly affect the proportions of transferable, unfertilized or degenerated oocytes/embryos. It was concluded that using low-dose X-sorted spermatozoa rather than normal-dose unsorted semen for the insemination of superovulated embryo donors can improve the proportion of transferable female embryos produced but this potential may not be achieved in commercial practice, particularly in cows, because of reduced fertilization rates when using low doses of X-sorted spermatozoa.     

Nuclear transfer of blastomeres expressing EGFP-reporter gene may improve the efficiency of transgenic cattle

The effect of timing of microinjection of DNA constructs on the efficiency of transgenic embryo production and improved efficiency and quality through combining EGFP as a reporter gene with nuclear transfer techniques were examined. From 12 to 24 h after insemination, constructs of pCXNeo-EGFP were microinjected into a pronucleus of bovine IVM-IVF zygotes. Due to the difficulty in visualizing pronuclei, the incidence of successful injection of linear DNA was higher when zygotes were injected between 20 and 24 h, as compared with an early period between 12 and 16 h after insemination. However, developmental competence of DNA-injected zygotes and the EGFP expression rate were not affected by the injection time. A majority of the embryos expressing EGFP signal were mosaic. Following nuclear transfer of blastomeres expressing EGFP, 4.5% of morulae that developed from the NT eggs had a strong EGFP signal in all live blastomeres. In other embryos, EGFP signal had been lost. When cells derived from the EGFP-positive NT morulae were subcultured, all the cells expressed strong EGFP signal at the second passage and demonstrated neomycin resistance. These results show that transient expression of nonintegrated EGFP appears frequently in EGFP-positive bovine embryos and that additional selection of EGFP-positive morulae after nuclear transfer of EGFP-positive blastomeres would facilitate selection of transgenic embryos.     

Factors affecting in vivo viability of DNA-injected bovine blastocysts produced in vitro

In vitro matured and fertilized bovine ova were microinjected with pBL1, which consisted of the bovine beta-casein gene promoter, human lactoferrin cDNA and SV40 polyadenylation signal. Of the 2931 zygotes injected, 2505 (85.5%) survived 1 h after DNA injection and were cultured in 50-microl drops of CR1aa medium containing 3 mg/ml BSA under mineral oil at 39 degrees C, 5% CO2 in air. Cleaved (2- to 8-cell) embryos were selected at approximately 48 h after DNA injection and then cultured further in 50-microl drops of CR1aa medium supplemented with 10% (v/v) FBS. Blastocysts were classified into 4 quality grades and 3 developmental stages by morphological criteria. Then all but poor quality blastocysts were nonsurgically transferred to the uterus of heifers 7 to 8 d after natural estrus. Following transfer, the recipients were observed for signs of estrus, and pregnancy was confirmed by palpation per rectum at approximately 60 d of gestation. Although 72.0% (1804/2505 ) of the DNA-injected zygotes reached 2- to 8-cell stages only 5.2% (131/2505) developed to blastocysts. A total of 75 DNA-injected, in vitro cultured blastocysts were transferred to 59 recipients. When 2 blastocysts were transferred to a single recipient, only the better quality embryo was counted. The overall pregnancy rate was 30.5% (18/59 ) and reflected 1) an apparent correlation between the quality of embryos and the pregnancy rate. However, the difference was not statistically significant. 2) expanded blastocysts had a higher pregnancy rate (50.0%, 11/22 ) than early (13.3%, 2 15 ) or mid (22.7%, 5/22 ) blastocysts with a significant difference between expanded and early blastocysts (P < 0.05). 3) the pregnancy rate of DNA-injected blastocysts was higher when they were transferred at Day 7 (34.5%, 10/29 ) or 8 (36.8%, 7/19 ) than at Day 6 (9.0%, 1/11 ). The results indicate that the developmental stage of DNA-injected bovine embryos may be one of contributing factors in improving the pregnancy rate after transfer, although the effects of the quality and culture period of the embryos may not be inconsequential.